ABSTRACT Aseptic encephalitis and meningitis are potentially fatal diseases defined by acute swelling of the brain or protective membranes covering the brain and spinal cord. These diseases can be caused by viruses, bacteria, fungi and parasites. Viruses are the most common cause of encephalitis, with >85% of all aseptic encephalitis caused by enterovirus. Human herpes viruses account for 4% of all viral infections, and West Nile virus is the most common of the arboviral infections in the U.S. According to the CDC, most cases of human enteroviral encephalitis resolve without complication. One the other hand, herpes simplex encephalitis results in rapid death in approximately 70% of cases if left untreated. Neisseria meningitides can be differentiated from other causes of meningitis by a rapidly spreading petechial rash, and other bacterial and fungal meningitis/encephalitis can readily be differentiated from viral causes by biochemistry and gram staining of the cerebrospinal fluid. Importantly, complications arising from CSF infection and appropriate treatment strategies depend on the type of infection and the species involved. For aseptic causes of the disease, case-adapted therapy is therefore dependent upon rapidly diagnosing and differentiating between viral infections. Nucleic acid amplification and detection assays have been considered the test of choice for CSF infections for more than a decade, yet there are only three FDA-approved viral diagnostic tests, each of which only detects one virus at a time. The objective of this project is therefore to develop and trial a "closed amplicon" gel element RT-PCR array for the diagnosis of encephalitis and meningitis. The assay is based on a set of real- time PCR and reverse transcriptase PCR assays developed by the Laboratory of Viral Diseases at the Wadsworth Center. The performance goal for Phase 1 is a closed amplicon RT-PCR array for the detection of enterovirus, herpes viruses (HSV-1, HSV-2, VZV, CMV, HHV-6), and West Nile virus, with an analytical LoD of d 50 genome copies per reaction. The assay will contain an exogenously spiked, quantified extraction and amplification control which is amplified and detected in the same multiplex reaction. Successful completion of either Specific Aim in Phase 1 will warrant a Phase 2 proposal focused on reagent stability, packaging and shelf life;pre-clinical experiments to demonstrate assay reproducibility, repeatability, sensitivity and specificity;and developing automated algorithms and decision rules for assay data analysis and reporting. PUBLIC HEALTH RELEVANCE: The closed-amplicon gel element microarray will be the first-of-its-kind combination of RT-PCR and microarray technology in a simple, inexpensive test kit for the diagnosis of aseptic encephalities and meningits. The underlying platform developed herein will likewise find broad application in many areas of infectious disease diagnostics.